Abstract
Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.
Highlights
Platelet lifespan is regulated by the intrinsic apoptotic pathway
ABT-737, the BH3 mimetic most well-characterised in platelets, inhibits Bcl-2 and Bcl-xL, triggering caspase activation, phosphatidylserine (PS) exposure in a Bak/Bax-dependent manner,[3] followed by release of PS-exposing extracellular vesicles (EVs) and secondary necrosis in vitro
This late timepoint was chosen as the BH3 mimetic most well-characterised in platelets, ABT-737, requires several hours of treatment to see the largest effects.[1,2,3,4,17]
Summary
Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL.[1,2] Such BH3 mimetics have become useful tools to understand the mechanisms that regulate platelet apoptosis. ABT-737, the BH3 mimetic most well-characterised in platelets, inhibits Bcl-2 and Bcl-xL, triggering caspase activation, phosphatidylserine (PS) exposure in a Bak/Bax-dependent manner,[3] followed by release of PS-exposing extracellular vesicles (EVs) and secondary necrosis in vitro. 4 The orally available analogue, ABT-263 (navitoclax), triggers platelet apoptosis and is associated with dose-limiting thrombocytopenia.[5] In contrast, ABT-199 (venetoclax), which is more selective for Bcl-2 over Bcl-xL, spares platelets and has been approved for treatment of chronic lymphocytic leukaemia.[6,7] It is important to understand which drugs trigger apoptosis in platelets
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